Current treatment options for pancreatic cancer (PDAC) are limited and ineffective. The recent genome-wide sequencing analysis of PDAC verified that mutation of the KRAS oncogene may be the most critical genetic alteration involved in PDAC growth. Thus, it is widely believed that anti-K-Ras targeted therapies will have a significant clinical benefit for the PDAC patient. While the development of direct anti-Ras therapies has proven to be difficult, there remains strong optimism that indirect targeting of Ras downstream effector signaling is our best hope for success. However, these efforts have been complicated by the fact that Ras utilizes multiple effector pathways to promote oncogenesis. A major focus of our previous proposal was to validate the role of specific effectors in K-Ras-driven PDAC growth. The most significant outcome of our studies is that we found that the RalGEF-Ral effector signaling network, rather than the more studied Raf-MEK-ERK mitogen-activated protein kinase cascade or phosphatidylinositol 3-kinase (PI3K)-AKT-mTOR effector pathways, may be the most critical signaling mechanism for the K-Ras-mediated tumorigenic, invasive and metastatic growth properties of PDAC cells. To date, Ral activation has been demonstrated only in PDAC patient tumors. Therefore, a key strength of our studies is the analysis of this pathway in the context of mutant KRAS and its role in more complex in vivo growth parameters of tumorigenic growth, invasion and metastasis in mouse models. Currently, the pharmaceutical industry has focused on the clinical development of inhibitors of the Raf- MEK-ERK and PI3K-AKT-mTOR pathways. We suggest that an effort should also be made to target the RalGEF-Ral pathway for PDAC treatment. The goal of our studies is a mechanistic dissection of the roles and mechanisms by which the biochemically-identical, yet biologically-distinct RalA and RalB isoforms promote PDAC tumorigenicity, invasion and metastasis. We expect that our findings will identify tractable targets for developing pharmacologic inhibitors of RalGEF-Ral signaling for PDAC treatment. Our studies will determine [1] the mechanisms of RalA-mediated anchorage-independent growth and tumorigencity in PDAC, [2] the mechanisms of RalB-mediated invasion and metastasis in PDAC, [3] the role of RalGEFs in K-Ras-mediated Ral activation in PDAC, and finally, [4] the roles of RalGEF and lesser studied effectors KRAS dependency, in regulation of gene expression and control of epithelial-mesenchymal transition. PUBLIC HEALTH RELEVANCE: The essentially 100% occurrence of RAS oncogene mutations in pancreatic cancer argues that anti-Ras inhibitors will be effective anti-cancer therapies for this deadline disease. In our studies we have identified a key signaling pathway that is crucial for pancreatic cancer tumor growth, invasion and metastasis. Therefore, we believe that our studies to better understand how this pathway, the RalGEF-Ral pathway, promotes Ras- driven cancer growth will lead to our identification of novel drugs for the effective treatment of pancreatic cancer.